Novel abietane diterpenoid compound, and composition comprising extract of torreya nucifera, or abietane diterpenoid compounds or terpenoid compounds isolated from them for prevention and treatment of cardiovascular disease

ABSTRACT

The present invention relates to a composition for the prevention and the treatment of cardiovascular disease containing extracts of  T. nucifera  or abietane diterpenoid compound or terpenoid compound isolated from the same as an effective ingredient. 
       T. nucifera  extracts or abietane diterpenoid compound or terpenoid compound isolated from the same of the present invention not only shows excellent anti-oxidative activity to LDL but also effectively inhibits ACAT activity. Further,  T. nucifera  extracts of the present invention reduce blood LDL cholesterol and total cholesterol. 
     Therefore, the composition of the present invention can be effectively used for the prevention and the treatment of cardiovascular diseases including hyperlipidemia and atherosclerosis caused by the LDL oxidation and the synthesis and accumulation of cholesteryl ester.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a Divisional of U.S. patent application Ser.No. 10/591,282, filed on Aug. 31, 2006, which in turn claims the benefitof priority from Korean Patent Application Nos. 10-2004-0014236,10-2004-0089372 and 10-2004-0112140 filed Mar. 3, 2004, Nov. 4, 2004 andDec. 24, 2004 respectively through PCT Application Serial No.PCT/KR2005/000472 filed Feb. 22, 2005 the contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel abietane diterpenoid compoundand a composition for the prevention and the treatment of cardiovasculardisease containing extracts of Torreya nucifera or abietane diterpenoidcompounds or terpenoid compounds isolated from the same as an effectiveingredient.

2. Description of Related Art

Cardiovascular diseases including atherosclerosis increase gradually asadult disease is growing. Atherosclerosis frequently occurs in cerebralartery or in coronary artery. Cerebral atherosclerosis carries headache,dizziness and mental disorder, and is a cause of cerebral infraction.Coronary arteriosclerosis causes pain and arrhythmia in the heart,leading to angina pectoris or myocardial infraction. Such diseasesfurther cause hypertension, heart disease, cerebral hemorrhage, etc,making atherosclerosis related diseases the leading cause of death ofmen at the age of 50˜60 s.

High blood cholesterol causes coronary cardiovascular diseases. In orderto reduce blood cholesterol, inhibition of enzyme involved in lipidmetabolism or a dietary treatment designed to limit the intake ofcholesterol and lipid has to be enforced.

For the purpose of preventing such diseases, attempts have been made toreduce low-density lipoprotein (LDL) by inhibiting cholesterolabsorption and biosynthesis thereof (Principles in Biochemistry, lipidbiosynthesis, 770-817, 3rd Edition, 2000 Worth Publishers, New York;Steinberg, D. et al. N. Engl. J. Med, 320: 915-924, 1989).

The production of LDL oxide in blood has been the subject of study sinceit is regarded as the cause of atherosclerosis (Circulation, 91:2488-2496, 1995; Arterioscler. Thromb. Vasc. Biol., 17: 3338-3346,1997). In particular, a recent report saying that the production of foamcells is resulted from the inflow of HM-LDL (highly modified LDL),generated by peroxidation and structural transformation of LDL, intomacrophages triggered the study on the mechanism of the production andelimination of LDL peroxide (Curr. Atheroscler. Res., 2: 363-372, 2000).

Plague formation and breakage in inside of vascular wall lead tomyocardial infraction, and chronic inflammation on vascular wall by thedamage of it results in atherosclerosis, which is believed to be arather defense mechanism than damage mechanism (Circ. Res. 89: 298-304,2001).

Acyl-CoA: cholesterol acyltransferase (ACAT) is an enzyme thatesterifies cholesterol and its working mechanism is in act in threeregions of body (intestine, liver and vascular wall cells).

First, ACAT esterifies cholesterol and then helps the absorption ofcholesterol in intestines. Second, cholesterol which is taken in fromoutside or produced in inside of body is accumulated in a carrier namedVLDL (very low-density lipoprotein) in the liver, which is then providedto each organ of body through blood vessels. At this time, cholesterolis converted into cholesteryl ester by ACAT, enabling the accumulationof cholesterol in a carrier. Third, ACAT esterifies cholesterol inarterial wall cells, promoting the accumulation of cholesterol in cells,which is a direct reason for atherosclerosis.

By the activity of ACAT, foam cells include a huge number of cholesterolester that is induced from cholesterol. Thus, the formation of foamcells induced from macrophages and smooth muscle cells is very importantin experimental and clinical aspects. The growth of foam cells invascular wall is directly related to the increase of ACAT activity.Therefore, an ACAT inhibitor might be effectively used as a powerfulanti-atherosclerotic agent.

Therefore, an ACAT activity inhibitor has to and is expected to (1)reduce cholesterol taken in by inhibiting the absorption of cholesterolin intestines, (2) reduce blood cholesterol by inhibiting the release ofcholesterol into blood vessels and (3) prevent atherosclerosis byinhibiting the accumulation of cholesterol in vascular wall cells.

All the ACAT activity inhibitors reported as of today are the inhibitorsof the activity of mouse liver microsomal ACAT or mouse liver macrophage(J774) ACAT. Human ACAT is divided into two types; ACAT-1 and ACAT-2.Human ACAT-1 (50 kDa) works largely in the liver, adrenal gland,macrophage and kidney of an adult, and human ACAT-2 (46 kDa) works inthe small intestine (Rudel, L. L. et al., Curr. Opin. Lipidol. 12:121-127, 2001). The inhibition of ACAT activity has been a usefulstrategy for the prevention and the treatment of hypercholesterolemia,cholesterol gallstones or atherosclerosis owing to its mechanisms ofinhibiting the absorption of cholesterol taken in from food andinhibiting the accumulation of cholesteryl ester in vascular wall(Buhman, K. K. et al., Nature Med. 6: 1341-1347, 2000).

Probucol, N,N′-diphenylenediamine, BHA (butylatedhydroxyanisol) and BHT(butylated hydroxy toluene), synthetic phenols used as anti-oxidatantagents, that have been used for the treatment of hyperlipidemia, reduceLDL cholesterol, weaken LDL-oxidation and reduce the lesion formation,showing excellent anti-oxidative activity but carrying serious sideeffects, so that they are limited in use.

Therefore, the treatment of patients with hyperlipidemia oratherosclerosis with a LDL anti-oxidative agent together with a lipidlowering agent is promising.

In the meantime, T. nucifera is a kind of evergreen needle-leaf talltree belonging to Taxaceae, which is only distributed in Korea andJapan. T. nucifera is an edible, ornamental, medicinal and industrialplant. Its seeds have been eaten or produced as oil. And, its fruitshave been used for the treatment of extermination, hair-regrowth,stomach-strengthening, and intestinal hemorrhage, especially in Chinesesmedicine and folk remedies, and the wood itself has been used forconstruction, making facilities and making ship. Ingredients of T.nucifera, separated from its leaves and seeds, are sesquiterpenoids(Sakai, T. et al., Bull. Chem. Soc. Japan, 38: 381, 1965), labdanes,abietanes including diterpenoids (Sayama, Y. et al, Agric. Bio. Chem.,35: 1068, 1971; Harrison, L. and Asakawa, Y., Phytochemistry, 26: 1211,1987) and flavonoids (Ahmad, I. et. al., Phytochemistry, 20: 1169,1981), etc.

The present inventors have searched a novel therapeutic agent forhyperlipidemia and atherosclerosis with less side effects, from naturalresources. And the present inventors have completed this invention byconfirming that T. nucifera extracts or abietane diterpenoid compoundsor terpenoid compounds isolated from the same has excellentanti-oxidative activity to LDL and inhibiting activity to ACAT enzyme aswell.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel abietanediterpenoid compound.

It is another object of the present invention to provide a compositionfor the prevention and the treatment of cardiovascular diseasecontaining extracts of T. nucifera or abietane diterpenoid compounds orterpenoid compounds isolated from the same as an effective ingredient.

In order to achieve the above object, the present invention provides anovel abietane diterpenoid compound and a composition for the preventionand the treatment of cardiovascular disease containing extracts of T.nucifera or abietane diterpenoid compounds or terpenoid compoundsisolated from the same as an effective ingredient.

The composition of the present invention includes pharmaceuticalcompositions, which are effective for the prevention and the treatmentof cardiovascular disease, and for health food compositions.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention is described in detail.

The present invention provides a novel abietane diterpenoid compoundrepresented by the following formula 1.

(R is dimetoxymethyl.)

The abietane diterpenoid compound of formula 1 is12-hydroxyabietic-8,11,13-trien-18-dimethylacetyl.

The present invention also provides a composition for the prevention andthe treatment of cardiovascular disease comprising either T. nuciferaextracts or abietane diterpenoid compounds represented by the followingformula 1 that are isolated from the extracts or terpenoid compoundsselected from a group consisting of compounds represented by thefollowing formula 2˜formula 5 that is also isolated from the extracts,as an effective ingredient.

(R is methyl, hydroxymethyl, aldehyde, methylester or dimethoxymethyl.)

Abietane diterpenoid compounds of the formula 1 include ferruginol(R=methyl), 18-hydroxyferruginol (R=hydroxymethyl), 18-oxoferruginol(R=aldehyde) and 12-hydroxyabietic-8,11,13-trien-18-oic acid methylester (R=methyl ester). The compounds of formula 2 and formula 3 areisopimaric acid and dehydroabietinol, respectively, both are abietanediterpenoid compounds, and the compound of formula 4 is kayadiol, alabdane diterpenoid compound. The compound of formula 5 is δ-cadinol, asesquiterpenoid compound.

The compounds of formula 1˜formula 5 can be used in the form ofpharmaceutically acceptable salts, in which all the salts, hydrates andsolvates that can be prepared by the conventional method are included.

T. nucifera extracts of the present invention are extracted from leaves,stems or seeds of T. nucifera by using water, alcohol or the mixturethereof. At this time, alcohol is preferably selected from a groupconsisting of methanol, ethanol and butanol.

Abietane diterpenoid compounds or terpenoid compounds are isolated fromT. nucifera extracts by the conventional method, and reagents on themarket can be used.

The extraction, separation and purification methods of T. nuciferaextracts or abietane diterpenoid compounds or terpenoid compoundsseparated from the extracts are described hereinafter.

Dried T. nucifera leaves (stems or seeds) are dipped in water, which areheated at 40˜120° C. for 2˜24 hours. The solution is filtered to obtainextracts and solid residues. The obtained extracts are concentratedunder reduced pressure to give hot water extracts of T. nucifera leaves(stems or seeds).

Or, dired T. nucifera leaves (stems or seeds) are dipped in methanol (orethanol) for 3 weeks and then the solution is filtered. Charcoal isadded to the filtrate, followed by stirring at room temperature for 12hours. The solution is filtered again and then concentrated, to whichwater is added to suspend the solution. The solution is then filteredonce again. The obtained upper layer is dissolved in ethyl acetate,which is then concentrated, resulting in oily yellow substances. Theconcentrated solution is dissolved in dichloromethane, to which n-hexaneis slowly added in order to perform recrystallization. The solution isfiltered with a filter-glass. The resultant liquid is concentrated,resulting in oily substances.

The prepared oil fraction was examined to measure its anti-oxidativeactivity to LDL and inhibiting effects on human ACAT-1 and -2, resultingin the confirmation that the oil fraction has dual inhibiting effectagainst LDL-oxidation and ACAT.

The obtained ethyl acetate oil fraction is separated by silica-gelcolumn chromatography by using the mixed solvents of n-hexane and ethylacetate as the mobile phases. At this time, the oil fraction isseparated into 17 fractions (fraction 1˜17) by using the mixed solventsof n-hexane and ethyl acetate as mobile phase solvents, and at thistime, the mixing ratios of n-hexane and ethyl acetate (EtOAc) are 98:2,97:3, 95:5, 10:1, 5:1, 3:1, 1:1 and EtOAc 100% (v/v).

Fraction 8 (593 mg) that shows the best anti-oxidative activity isseparated by silica-gel column chromatography by using the mixedsolvents of n-hexane and ethyl acetate as mobile phases. At this time,as the mobile phase solvents, n-hexane is mixed with EtOAc at the ratiosof 98:2, 95:5, 10:1, 5:1, 3:1, 1:1 and EtOAc 100% (v/v), resulting in 11fractions (fraction 8-1˜8-17). Among 11 fractions, fraction 8-8˜8-10(n-hexane:EtOAc 5:1˜1:1, 149 mg), that show the high anti-oxidativeactivities, are mixed, and the mixture is purified by preparative TLC(CHCl₃/MeOH=80-1) and sephadex LH-20 column (CHCl₃/MeOH=1:1) to give twopurified compounds. These compounds are12-hydroxyabietic-8,11,13-trien-18-dimethylacetal (R=dimetoxymethyl, 18mg) and 12-hydroxyabietic-8,11,13-trien-18-oic acid methylester(R=methylester, 17.5 mg) of formula 1.

Ethyl acetate oil fraction obtained above is separated by silica-gelcolumn chromatography by using the mixed solvents of n-hexane and ethylacetate as the mobile phases. At this time, the mobile phase solventsare preferably ethyl acetate:n-hexane=10˜20:90˜80 (v/v). Among compoundsof formula 1, which are pure active ingredients, ferruginol (R=methyl,22 mg), 18-hydroxyferruginol (R=hydroxymethyl, 307 mg) and18-oxoferruginol (R=aldehyde, 62 mg) are obtained from 1 kg of dried T.nucifera leaves by the above method.

Ethyl acetate oil fraction obtained above is separated by silica-gelcolumn chromatography by using the mixed solvents of ethyl acetate andn-hexane as the mobile phases. The mobile phase solvents are prepared bymixing n-hexane and ethyl acetate at the ratio of 10:1, 5:1, 3:1, 1:1(v/v) and EtOAc 100%, resulting in the separation of 11 fractions(fraction 1˜11).

Fraction 5 showing a very strong ACAT inhibiting activity is separatedby silica-gel column chromatography by using the mixed solvent ofn-hexane and ethyl acetate as a mobile phase. At this time, the mobilephase solvent is prepared as n-hexane:ethyl acetate=7:1 (v/v) toseparate 7 fractions (fraction 5-1˜5-7) from fraction 5. Fraction 5-4having excellent ACAT inhibiting activity is separated by silica-gelcolumn chromatography. And, 4 fractions (fraction 5-4˜5-4-4) areseparated therefrom by using chloroform 100% as a mobile phase. From thefraction 5-4-1, the compound of formula 2 (isopimaric acid, 76 mg), apure active compound, is separated.

14 fractions (fraction 10-1˜10-14) are separared from fraction 10,prepared from the first column, by silica-gel column chromatographyusing methylene chloride:methanol=50:1 (v/v) as a mobile phase solvent.And, 11 fractions (fraction 10-4-1˜10-4-11) are separated from theactive fraction 10-4 by C18 reversed phase column chromatography usingmethanol:water=15:1 as a mobile phase solvent. 5 fractions(10-4-5˜10-4-5-5) are separated from the fraction 10-4-5 having strongACAT inhibiting activity by silica-gel column chromatography using themixed solvents of n-hexane and ethyl acetate which are prepared at theratios of 50:1, 30:1, 10:1, 1:1 (v/v) and EtOAc 100% as the mobile phasesolvents. A pure active compound represented by formula 3(dehydroabietinol, 25 mg) is obtained from the fraction 10-4-5-2.

A pure active compound of formula 4 (kayadiol, 40 mg) is given from thefraction 11 obtained from the first column by using recrystallizationsolvent n-hexane:ethyl acetate=5:1 (v/v).

12 fractions (fraction 10-6-1˜10-6-12) are separared from fraction 10-6,one of active fraction among 14 fractions obtained from the secondcolumn, by C18 reversed phase column chromatography usingmethanol:water=10:1 (v/v) as a mobile phase solvent. Silica-gel columnchromatography is also performed with active fraction 10-6-3 usingn-hexane:ethyl acetate=15:1, 10:1, 5:1, 1:1 and EtOAc 100% as the mobilephase solvents, resulting in 5 fractions (fraction 10-6-3-1˜10-6-3-5).Finally, a pure active compound of formula 5 (δ-cadinol, 15 mg) isprepared from the fraction 10-6-3-1.

T. nucifera extracts of the present invention or abietane diterpenoidcompound isolated from the same has low IC₅₀ value, indicating that ithas excellent anti-oxidative activity to LDL.

T. nucifera extracts of the present invention or abietane diterpenoidcompound or terpenoid compound isolated from the same show excellentACAT inhibiting activity in hACAT-1 and in hACAT-2.

T. nucifera extracts of the present invention also reduce serum LDL andblood cholesterol.

Therefore, the composition of the present invention can be effectivelyused for the prevention and the treatment of cardiovascular diseasessuch as hyperlipidemia and atherosclerosis which are caused by thesynthesis and the accumulation of cholesteryl ester.

The composition of the present invention can additionally include, inaddition to T. nucifera extracts or abietane diterpenoid compound orterpenoid compound isolated from the same, one or more effectiveingredients having same or similar function to the extract or compoundsseparared therefrom.

The composition of the present invention can also include, in additionto the above-mentioned effective ingredients, one or morepharmaceutically acceptable carriers for the administration.Pharmaceutically acceptable carriers can be selected or be prepared bymixing more than one ingredients selected from a group consisting ofsaline, sterilized water, Ringer's solution, buffered saline, dextrosesolution, maltodextrose solution, glycerol and ethanol. Other generaladditives such as anti-oxidative agent, buffer solution, bacteriostaticagent, etc, can be added. In order to prepare injectable solutions,pills, capsules, granules or tablets, diluents, dispersing agents,surfactants, binders and lubricants can be additionally added. Thecomposition of the present invention can further be prepared in suitableforms for each diseases or according to ingredients by following themethod represented in Remington's Pharmaceutical Science (the newestedition), Mack Publishing Company, Easton Pa.

The composition of the present invention can be administered orally orparenterally (for example, intravenous, hypodermic, local or peritonealinjection). The effective dosage of the composition can be determinedaccording to weight, age, gender, health condition, diet, administrationfrequency, administration method, excretion and severity of a disease.The dosage of T. nucifera extracts is 10˜2,000 mg/kg per day, andpreferably 50˜500 mg/kg per day. The dosage of the compounds of formula1 formula 5 is 0.1˜100 mg/kg per day and preferably 0.5˜10 mg/kg perday. Administration frequency is once a day or preferably a few times aday.

T. nucifera extracts of the present invention or abietane diterpenoidcompound or terpenoid compound isolated from the same was orallyadministered to mice to investigate toxicity. As a result, it wasevaluated to be safe substance since its estimated LD₅₀ value is muchgreater than 1,000 mg/kg in mice.

The composition of the present invention can be administered singly ortreated along with surgical operation, hormone therapy, chemotherapy andbiological reaction regulator, to prevent and treat cardiovasculardiseases.

The composition of the present invention can be included in health foodsfor the purpose of improving cardiovascular diseases. At this time, T.nucifera extracts of the present invention or abietane diterpenoidcompound or terpenoid compound isolated from the same can be added as itis or after being mixed with other food or ingredients, according to theconventional method. The mixing ratio of effective ingredients isdetermined by the purpose of use (prevention, health or therapeutictreatment). In the case of producing food or beverages containing T.nucifera extracts of the present invention or abietane diterpenoidcompound or terpenoid compound isolated from the same, it is preferablyadded by 120 weight %, more preferably 510 weight %, to the rawmaterial. However, the content of the extract might be less than theabove when it is administered for long-term to improve health conditionsbut the effective dosage could contain more than the above amountbecause the extract of the invention is very safe.

There is no limit in applicable food, which is exemplified by meat,sausages, bread, chocolate, candies, snacks, cookies, pizza, ramyun,noodles, dairy products including ice cream, soups, beverages, tea,drinks, alcoholic drinks and vitamin complex, etc, and in fact everyhealth food generally produced are all included.

Health beverages containing the composition of the present invention canadditionally include various flavors or natural carbohydrates, etc, likeother beverages. The natural carbohydrates above can be one ofmonosaccharides such as glucose and fructose, disaccharides such asmaltose and sucrose, polysaccharides such as dextrin and cyclodextrin,and sugar alcohols such as xylitol, sorbitol and erythritol. As asweetener, either natural sweetener such as thaumatin and stevia extractor artificial sweetener such as saccharin and aspartame can be used. Theratio of natural carbohydrate to the composition of the presentinvention is preferably 0.01˜0.04 g to 100 ml, more preferably 0.02˜0.03g to 100 ml.

In addition to the ingredients mentioned above, the composition of thepresent invention can include in variety of nutrients, vitamines,electrolytes, flavoring agents, colouring agents, pectic acid and itssalts, arginic acid and its salts, organic acid, protective colloidalviscosifiers, pH regulators, stabilizers, antiseptics, glycerin,alcohols, carbonators which used to be added to soda, etc. Thecomposition of the present invention can also include natural fruitjuice, fruit beverages and fruit flesh addable to vegetable beverages.All the mentioned ingredients can be added singly or together. Themixing ratio of those ingredients does not matter in fact, but ingeneral, each can be added by 0.01˜0.1 weight part per 100 weight partof the composition of the invention.

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

EXAMPLE 1 Extraction, Isolation and Purification of Abietane DiterpenoidCompound from T. nucifera

1. Preparation of Hot Water Extracts from T. nucifera Leaves

1 kg of T. nucifera leaves purchased in Jeju island, Korea, was washedand dried. 1000 ml of water was added to 250 g of the dried T. nuciferaleaves, which was boiled at 90° C. for 4 hours. The solution wasfiltered, resulting in extracts and solid phase residues. The obtainedextracts were concentrated under reduced pressure to give 30 g of hotwater extracts of T. nucifera leaves.

2. Preparation of Hot Water Extracts from T. nucifera Seeds

Hard shells of T. nucifera seeds were removed and then the seeds werepulverized by using a pulverizer. 500 ml of water was added to 120 g ofcrushed T. nucifera seeds, which was boiled at 90° C. for 4 hours. Thesolution was filtered, resulting in extracts and solid phase residues.The obtained extracts were concentrated under reduced pressure to give 9g of hot water extracts of T. nucifera seeds.

3. Preparation of Alcohol Extracts from T. nucifera Leaves

2.16 kg of T. nucifera leaves purchased in Jeju island, Korea, waswashed and dried. The dried T. nucifera leaves were put in 18 l of 100%ethanol, which was left at room temperature for 3 weeks. The solutionwas filtered with a filter paper. Charcoal was added to the filtrates,followed by stirring at room temperature for 12 hours. The solution wasfiltered and concentrated under reduced pressure to give an oily yellowsubject.

4. Isolation and Purification of Abietane Diterpenoid Compounds from T.nucifera Extracts

The oily yellow subject obtained from the above 3 was suspended in 1,000ml of water, then fractionized with n-hexane, chloroform and ethylacetate in that order. As a result, 75 g of n-hexane soluble extracts,37 g of chloroform soluble extracts and 18 g of ethyl acetate solubleextracts were obtained. The oily yellow subject obtained from the ethylacetate layer has excellent anti-oxidative effect on LDL.

Silica-gel column chromatography (silica-gel: Merck, Art, 9385, columnsize: φ 7×40 cm) was performed with 18 g of ethyl acetate oil fractionobtained above by using the mixed solvents of n-hexane and ethyl acetateas the mobile phases. At this time, the mixed solvents used as mobilephases was developed by 15 l respectively as n-hexane:EtOAc=98:2, 97:3,95:5, 10:1, 5:1, 3:1, 1:1 and EtOAc 100% (v/v) to separate 17 fractions(fraction 1˜17), and anti-oxidative activity of each fraction wasinvestigated.

Fraction 8 (593 mg) that showed the best anti-oxidative activity wasseparated by silica-gel column chromatography by using the mixedsolvents of n-hexane and ethyl acetate as mobile phases. At this time,the mobile phase solvents were developed as n-hexane:EtOAc=98.2, 95:5,10:1, 5:1, 3:1, 1:1 and EtOAc 100% (v/v), by 100 ml each, resulting in11 fractions (fraction 8-1˜8-17). Anti-oxidative activity of eachfraction was investigated. And the mixture of fractions 8-8˜8-10(n-hexane:EtOAc 5:1˜1:1, 149 mg), each of which had the most excellentanti-oxidative activity, was purified by preparative TLC (Silica-gel60F₂₅₄, Merck, Art. 5744, CHCl₃/MeOH=80:1) and sephadex LH-20 columnchromatography (Sigma-Aldrich Co., USA, CHCl₃/MeOH=1:1), resulting intwo purified compounds, that were two of those compounds represented byformula 1 (18 mg, 17.5 mg).

5. Analyses of the Structures of Abietane Diterpenoid Compounds

The molecular weight and formula of the compound obtained in the above 4were investigated by using VG high resolution SC/MS spectrometer(Election Ionization MS, Autospec-Ultima), and polarity of it was alsomeasured with a polarimeter (Jasco DIP-181 digital polarimeter). The NMRanalyses were performed (Bruker AMX 300, 500) to obtain ¹H NMR, ¹³C NMR,HOMO-COSY, HMQC (¹H-Detected heteronuclear Multiple-Quantum Coherence),HMEC (Heteronuclear Multiple-Bond Coherence) and DEPT (DistortionlessEnhancement by Polarization) spectra and molecular structure of thecompound was determined.

The results are shown in below. After comparing with the other resultsshown in earlier reports, one of the compounds represented by formula 1was confirmed to be 12-hydroxyabietic-8,11,13-trien-18-dimethylacetal,which is a novel compound not reported yet. The other compound wasconfirmed to be 12-hydroxyabietic-8,11,13-trien-18-oic acid methylester.

(R is dimethoxymethyl.)

1) Physical property: yellow oil

2) Polarity: [α]_(D) ²⁵-5.8° (c=0.3, CHCl₃)

3) Molecular weight: 346

4) Molecular formula: C₂₂H₃₄O₃

5) ¹H NMR (CDCl₃, 500 MHz) δ 0.9 (s, 3H, H-19), 1.12 (s, 3H, H-20), 1.15(d, J=7.2 Hz, H-16), 1.16 (d, J=7.2 Hz, H 17), 1.27 (dt, T=4.2, 12.7 Hz,H-1a), 1.36-1.43 (m, 2H, H-3), 1.53-1.62 (m, 2H, H-2), 1.64-1.75 (m, 2H,H-6), 1.81 (dd, J=1.7, 12.1 Hz, H-5), 2.05 (d like, J=12.6 Hz, H-1b),6.55 (s, 1H, H-11), 6.75 (s, 1H, H-14).

6) ¹³C NMR (CDCl₃, 125 MHz) δ 16.7 (C-19), 18.3 (C-2), 19.4 (C-6), 22.5(C-16), 22.7 (C-17), 25.2 (C-20), 26.7 (C-15), 29.3 (C-7), 30.4 (C-3),37.3 (C-10), 38.2 (C-1), 42.6 (C-4), 42.8 (C-5), 58.7 (C-18b), 59.0(C-18a), 110.9 (C-11), 113.3 (C-18), 126.5 (C-14), 126.9 (C-8), 131.4(C-13), 148.7 (C-9), 150.7 (C-12).

7) EIMS (rel. int.) m/z [M]³⁰ 59 (22%), 75 (100%), 189 (14%), 201 (10%),346 (33%).

[12-hydroxyabietic-8,11,13-trien-18-oic acid methylester]

(R is methylester.)

1) Physical property: yellow oil

2) Polarity: [α]_(D) ²⁵+75.7° (c 0.28, EtOH)

3) Molecular weight: 330

4) Molecular formula: C₂₁H₃₀O₃

5) ¹H NMR (CDCl₃, 500 MHz) δ 1.12 (s, 3H, H-20), 1.15 (d, J=7.4 Hz, 3H,H-16), 1.16 (d, J=7.3 Hz, 3H, H-17), 1.19 (s, 3H, H-19), 1.29 (m, 1H,H-6a), 1.41 (m, 1H, H-2a), 1.55-1.77 (m, 5H, H-1α, H-2β, H-3, H-6β),2.12 (d like, J=12.7 Hz, 1H, H-1β), 2.14 (dd, J=1.7, 12.5 Hz, 1H, H-5)2.74 (m, 2H, H-7), 3.04 (m, 1H, H-15), 3.59 (s, 3H, CO₂Me) 4.58 (s,—OH), 6.55 (s, 1H, H-11), 6.74 (s, 1H, H-14).

6) ¹³C NMR (CDCl₃, 125 MHz) δ 16.5 (C-19), 18.5 (C-2), 21.8 (C-6), 22.5(C-16), 22.7 (C-17), 25.0 (C-20), 26.8 (C-15), 29.2 (C-7), 36.6 (C-1),36.9 (C-4), 38.0 (C-3), 44.8 (C-5), 47.7 (C-10), 51.9 (—OMe), 110.8(C-11), 126.7 (C-14), 127.0 (C-8), 131.7 (C-13), 147.9 (C-9), 150.8(C-12), 179.2 (C-18)<

EXAMPLE 2 Extraction, Isolation and Purification of Abietane DiterpenoidCompounds from T. nucifera

1. Preparation of Alcohol Extracts from T. nucifera Leaves

1 kg of T. nucifera leaves purchased in Jeju island, Korea, was washedand dried. The dried T. nucifera leaves were put in 4 l of 95% methanol,which was left at room temperature for 3 weeks. The solution wasfiltered with a filter paper. Charcoal was added to the filtrates,followed by stirring at room temperature for 12 hours. The solution wasfiltered and concentrated under reduced pressure to give an oily yellowsubject.

2. Isolation and Purification of Abietane Diterpenoid Compounds from T.nucifera Extracts

The oily yellow substances obtained in the above 1 was suspended in 200ml of water, which was then filtered with a filter paper. The upperlayer was dissolved in ethyl acetate. The solution was concentrated togive 40 g of oily yellow subject. The concentrated solution wasdissolved in dichloromethane, to which n-hexane was slowly added,resulting in recrystallization. After filtering with a filter-glass, theliquid phase was concentrated to give 30 g of oily subject.

Anti-oxidative activity to LDL and human ACAT-1 and -2 inhibitingcapacity of the obtained oily fraction were investigated, resulting inthe confirmation of dual inhibiting effects.

Silica-gel column chromatography (silica-gel: Merck, Art. 9385, columnsize: φ7×40 cm) was performed with 30 g of the oily fraction, obtainedabove, by using the mixed solvent of n-hexane and ethyl acetate as amobile phase to separate fractions therefrom. When ethylacetate:n-hexane=1:9 (v/v) was used as a mobile phase solvent, an activesubstance was separated best, from which pure active ingredientsferruginol (R methyl, 22 mg), 18-hydroxyferruginol (R=hydroxymethyl, 307mg) and 18-oxoferruginol (R=aldehyde, 62 mg) were obtained.

100 mg of 18-hydroxyferruginol (R=hydroxymethyl), a colorless solidcompound, was dissolved in 5 ml of dichloromethane, to which 10 ml ofn-hexane was slowly added. To the solution was added 1 ml ofdiethylether, which was left at room temperature for 24 hours. Theresultant single crystal was identified by X-ray spectroscopy.

3. Analyses of the Structures of Abietane Diterpenoid Compounds

The results of analyses of the structures of the products obtained inthe above 2 are shown in below, that is, the compounds of formula 2 wereconfirmed to be ferruginol (R=methyl), 18-hydroxyferruginol(R=hydroxymethyl) and 18-oxoferruginol (R=aldehyde) [L. J. Harrison andY. Asakawa, Pytochemistry, 1987, 26, 1211].

(R is methyl.)

1) Physical property: colorless oil

2) Polarity: [α]_(D) ²⁵ ⁺56.6° (c=0.6, CHCl₃)

3) Molecular weight: 286

4) Molecular formula: C₂₀H₃O

5) ¹H-NMR (CDCl₃, 300 MHz) δ 0.90 (s, 3H, H-18), 0.93 (s, 3H, H-19),1.16 (s, 3H, H-20), 1.21 (d, J=5.1 Hz, 3H, H-16), 1.23 (d, J=5.1 Hz, 3H,H-17), 1.20 (dd, J=3.0, 10.4 Hz, 1H), 1.22 (t like, J=5.1 Hz, 6H, H-18,19), 1.31 (dd, J=1.7, 9.3 Hz, 1H), 1.38 (dd, J=2.8, 10.4 Hz, 1H),1.58-1.89 (m, 3H), 1.84 (m, 1H), 2.15 (dd like, J=0.7, 8.7 Hz, 1H), 2.76(ddd, J=1.3, 5.3, 8.5 Hz, 1H, H-7a), 2.85 (ddd, J=1.3, 5.2, 8.6 Hz, 1H,H-7b), 3.10 (m, 1H, H-15), 4.49 (br, 1H, —OH), 6.62 (s, 1H, H-13), 6.82(s, 1H, H-10).

6) ¹³C-NMR (CDCl₃, 75 MS) δ 19.2 (C-2), 19.3 (C-6), 21.6 (C-19), 22.5(C-16), 22.7 (C-17), 24.8 (C-20), 26.8 (C-15), 29.7 (C-7), 33.3 (C-18),33.4 (C-4), 37.5 (C-10), 38.8 (C-1), 41.7 (C3), 50.3 (C-5), 110.9(C-11), 126.6 (C-14), 127.3 (C-8), 131.3 (C-13), 148.7 (C-9), 150.6(C-12).

7) EIMS (rel. int.) m/z [M]+ 69 (78.4), (64.5), 159.1 (47.5), 175.1(90.7), 187.1 (63.8), 201.1 (86.0), 215.1 (54.4), 229.2 (56.5), 271.2(100), 286.2 (99.6).

(R is hydroxymethyl.)

1) Physical property: colorless prizm, M.P.=185˜187° C.

2) Polarity: [α]_(D) ²⁵ 110° (c=0.2, CHCl₃)

3) Molecular weight: 302

4) Molecular formula: C₂₀H₃₀O₂

5) ¹H NMR (MeOD, 500 MHz) δ 0.84 (s, 3H, H-19), 1.16 (t like, J=5.7 Hz,6H, H-16, 17), 1.18 (s, 3H, H-20), 1.31 (m, 2H, H-3), 1.51 (dt, J=3.9,13.4 Hz, 1H, H-2a), 1.65 (m, 3H, H-6, H-2b), 1.79 (m, 2H, H-1), 2.20 (dlike, J=12.7 Hz, 1H, H-5), 2.75 (d like, J=7.7 Hz, 2H, H-7), 3.09 (d, J11.0 Hz, 1H, H-18a), 3.22 (m, 1H, H-15), 3.30 (s, 1H, —OH), 3.41 (d,J=11.0 Hz, 1H, H-18b), 6.63 (s, 1H, H-11), 6.78 (s, 1H, H-14).

6) ¹³C-NMR (MeOD, 125 MHz) δ 18.0 (C-19), 19.8 (C-2), 20.1 (C-6), 23.2(C-16, 17), 25.8 (C-20), 27.7 (C-15), 30.4 (C-7), 36.3 (C-3), 38.4(C-4), 38.9 (C-10), 39.9 (C-1), 45.0 (C-5), 72.0 (C-18), 111.6 (C-11),126.9 (C-14), 127.2 (C-8), 133.2 (C-13), 149.1 (C-9), 153.1 (C-12).

7) EIMS (rel. int.) m/z [M]+ 147 (46.5), 175 (70.2), 189 (78.0), 201(45.0), 227 (50.7), 269 (100.0), 287 (60.0), 302 (95.1).

(R is aldehyde.)

1) Physical property: colorless prizm, M.P.=140˜142° C.

2) Polarity: [α]_(D) ²⁵ ⁺61° (c=0.2, CHCl₃)

3) Molecular weight: 300

4) molecular formula: C₂₀H₂₈O₂

5) ¹H-NMR (CDCl₃3, 300 MHz) δ 1.13 (s, 3H, H-19), 1.21 (s, 3H, H-20),1.22 (d, J=4.23 Hz, 6H, 3H-16, 17), 1.28 (m, 2H), 1.44 (m, 3H), 1.79 (m,3H), 2.22 (m, 1H), 2.79 (m, 2H, H-7), 3.09 (m, 1H, H-15), 4.52 (s, 1H,C12-OH), 6.62 (s, 1H, H-11), 6.82 (s, 1H, H-14), 9.24 (s, 1H, —CHO).

6) ¹³C-NMR (CDCl₃, 75 MHz) δ 14.0 (C-19), 17.8 (C-2), 21.5 (C-6), 22.5(C-16), 22.7 (C-17), 25.0 (CO₂₀), 26.8 (C-15), 29.0 (C-7), 32.0 (C-3),36.2 (C-10), 37.8 (C-1), 42.8 (C-5), 49.8 (C-4), 110.8 (C-11), 126.7(C-8), 126.9 (C-14), 132.0 (C-13), 147.2 (C-9), 150.9 (C-12), 206.4(C18).

EXAMPLE 3 Extraction, Isolation and Purification of Terpenoid Compoundsfrom T. nucifera

1. Extraction, Isolation and Purification of Compounds from T. nucifera

1 kg of T. nucifera leaves purchased in Jeju island, Korea, was washedand dried. The dried T. nucifera leaves were dipped in 4 l of 100%methanol, which was left at room temperature for 3 weeks. The solutionwas filtered with a filter paper, and charcoal (100 g) was addedthereto, followed by stirring at room temperature for 12 hours. Thesolution was filtered and then concentrated under reduced pressure togive an oily yellow substances. The product was suspended in 200 ml ofwater, followed by filtering with a filter paper. The upper layer wasdissolved in ethyl acetate, which was concentrated to give 40 g of oilyyellow substances. The concentrated solution obtained above wasdissolved in dichloromethane, to which n-hexane was added slowly,resulting in recrystallization. After filtering with a filter glass, theliquid phase was concentrated to give 30 g of the oily substances. Theoily fractions obtained above have excellent hACAT-1 and hACAT-2inhibiting activities.

Silica-gel column chromatography (Silica-gel: Merck, Art. 9385, Columnsize: φ7×40 cm) was performed with 16 g of the above oily fraction byusing the mixed solvents of ethyl acetate and n-hexane as mobile phases.At this time, the mixed solvents used as mobile phases was developed by1000 ml respectively as n-hexane:EtOAc=10:1, 5:1, 3:1, 1:1 and EtOAc100% (v/v) to isolate 11 fractions (fraction 1˜11) Fraction 5 (6 g)showing high ACAT inhibiting activity was separated by silica-gel columnchromatography (Column size: φ4×20 cm) by using the mixed solvent ofn-hexane and ethyl acetate as a mobile phase. At this time, the mixedsolvent used as a mobile phase was developed as n-hexane:EtOAc 7:1 (v/v)to isolate 7 fractions (fraction 5-1˜5-7) (250 ml each). Fraction 5-4(100 mg) showing excellent ACAT inhibiting activity was also separatedby silica-gel column chromatography (Column size: φ2.5×10 cm). Thesolution was developed by using chloroform 100% as a mobile phasesolvent, as a result, 4 fractions (fraction 5-4-1˜5-4-4) were separated(50 ml each). And, a pure active compound of formula 2 was obtained (25mg) from the fraction 5-4-1.

Silica-gel column chromatography (Column size: φ1.5×20 cm) was performedwith fraction 10 (1.34 g) obtained from the first column by mobile phasesolvent of methylenechloride:methanol=50:1 (v/v), resulting in theisolation of 14 fractions (fraction 10-1˜10-14) (50 ml each). At thistime, active fraction 10-4 (100 mg) was developed by usingmethanol:water=15:1 as a mobile phase solvent, followed by C18 reversedphase column chromatography (Column size: φ1×10 cm). As a result, 11fractions (45 ml each) were isolated. Silica gel column chromatography(Column size: φ1×8 cm) was performed with the fraction 10-4-5 havingstrong inhibiting activity by using the mixed solvents of n-hexane andethyl acetate as mobile phases. At this time, the mixed solvents wereprepared at the ratios of n-hexane:EtOAc=50:1, 30:1, 10:1, 1:1 (v/v) andEtOAc 100%. As a result, 5 fractions (10-4-5-1˜10-4-5-5) were separated(30 ml each). A pure active compound of formula 3 (76 mg) was obtainedfrom the fraction 10-4-5-2.

A compound of formula 4 (40 mg) was obtained by using a recrystallizingsolvent n-hexane:EtOAc=5:1 (v/v) from the fraction 11 (149 mg) obtainedfrom the first column.

C18 reversed phase column chromatography (Column size: φ1.5×13 cm) wasperformed with fraction 10-6 (85 mg), which was comparatively activeamong 14 fractions obtained from the second column, by developing themobile phase solvent of methanol:water=10:1 (v/v), resulting in theseparation of 12 fractions (fraction 10-6-1˜10-6-12) (45 ml each). Amongthose fractions, one of active fraction 10-6-3 (40 mg) was separated bysilica-gel column chromatography (Column size: φ1×7 cm) by developingthe mixed solvents of n-hexane:EtOAc=15:1, 10:1, 5:1, 1:1 (v/v) andEtOAc 100%. As a result, 5 fractions (10-6-3-1˜10-6-3-5) were separated(30 ml each). And a pure active compound of formula 5 (15 mg) wasobtained from the fraction 10-6-3-1.

2. Analyses of the Structures of Terpenoid Compounds

The structures of the compounds obtained in the above 1, wasinvestigated. As a result, the compound of formula 2 was identified asisopimaric acid [Y.-H. Kuo and W.-C. Chen, J. Chin. Chem. Soc., 46: 819,1999], the compound of formula 3 was identified as dehydroabietinol [H.L. Ziegler et al., Planta Med., 68: 547, 2002], the compound of formula4 was identified as kayadiol [J. D. P. Teresa et al., Argic. Biol.Chem., 35: 1068, 1971] and the compound of formula 5 was identified asδ-cadinol [Bull. Chem. Soc. Jpn., 137: 1053, 1963].

1) Physical property: Coloress prizm, M.P.=185˜187° C.

2) Polarity: [α]_(D) ²⁵ +10.5° (c=0.42, EtOH)

3) Molecular weight: 302

4) Molecular formula: C₂₀H₃₀O₂

5) ¹H-NMR (CDCl₃, 500 MHz) δ 0.86 (s, 3H, H-19), 0.91 (s, 3H, H-20),1.12 (m, 1H), 1.27 (s, 3H, H-17), 1.37 (m, 2H), 1.48 (m, 1H), 1.55 (m,3H), 1.67-2.03 (m, 9H), 4.87 (dd, J=1.7, 10.8 Hz, 1H, H-16ab, H-16ax),4.93 (dd, J=0.6, 17.5 Hz, 1H, H-16ba, H-16bx), 5.32 (d like, J=4.1 Hz,1H, H-7), 5.80 (dd, J=10.8, 17.6 Hz, H-15ax, H-15bx), 12.1 (brs, 1H,—COOH).

6) ¹³C-NMR(CDCl₃, 125 MHz) δ 15.3 (C-20), 17.1 (C-19) 17.9 (C-2), 20.0(C-17), 25.2 (C-6), 35.0 (C-10), 36.1 (C-12), 36.8 (C-3), 37.0 (C-13),38.8 (C-1), 45.0 (C-5), 46.1 (C-4), 46.3 (C-14), 52.0 (C-9), 109.3(C-16), 121.0 (C-7) 135.7 (C-8), 150.3 (C-15), 185.6 (C-18).

7) EIMS (rel. int.) m/z [M]⁺ 105 (40.7), 187 (36.9), 241 (47.8), 257(30.9), 273 (26.9), 287 (47.1), 302 (100.0).

1) Physical property: Viscous oil

2) Polarity: [α]_(D) ²⁵ +50° (c=0.24, CHCl₃)

3) Molecular weight: 286

4) Molecular formula: C₂₀H₃₀O

5) ¹H-NMR (CDCl₃, 300 MHz) δ 0.90 (s, 3H, H-19), 1.22 (s, 3H, H-20),1.23 (t like, J=3.6 Hz, 6H, H-16, 17), 1.34-1.50 (m, 3H, H-1a, H-3),1.63-1.82 (m, 5H, H-2, 5, 6), 2.29 (d like, J=12.6 Hz, 1H, H-1β),2.79-2.92 (m, 3H, H-7, 15), 3.24 (d, J=11.11 Hz, 1H, H-18a), 3.48 (d,J=11.1 Hz, 1H, H-18β), 6.89 (s, 1H, H-14), 6.99 (d, J=8.1 Hz, 1H, H-12),7.19 (d, J=7.8 Hz, 1H, H-11).

6) ¹³C-NMR (CDCl₃, 75 MHz) δ 17.4 (C-19), 18.6 (C-2), 18.8 (C-6), 24.0(C-16, 17), 25.3 (C-20), 30.1 (C-7), 33.4 (C-15), 35.1 (C-3), 37.3(C-10), 37.8 (C-4), 38.4 (C-1), 43.9 (C-5), 72.2 (C-18), 123.8 (C-12),124.2 (C-11), 126.8 (C-14), 134.7 (C-8), 145.5 (C-13), 147.3 (C-9).

7) EIMS (rel. int.) m/z [M]⁺ 159 (47.5), 173 (60.9), 185 (27.0), 253(100), 271 (87.2), 286 (32.6).

1) Physical property: Amorphous powder

2) Polarity: [α]_(D) ²⁵ +18.4°(c=0.3, CHCl₃)

3) Molecular weight: 306

4) Molecular formula: C₂₀H₃₄O₂

5) ¹H-NMR (CDCl₃, 5000 MHz) δ 0.72 (s, 3H, H-20), 0.75 (s, 3H, H-18),1.02 (dt, J=4.2, 12.7 Hz, 1H), 1.28 (m, 1H), 1.35 (dt, J=4.2, 12.7 Hz,1H), 1.37-1.48 (m, 4H), 1.55-1.65 (m, 5H), 1.67 (s, 3H, H-16), 1.76-1.84(m, 2H), 2.00 (dt, J=4.6, 12.6 Hz, 1H), 2.38 (m, 1H), 3.10 (d, J=10.9Hz, 1H, H-19a), 3.42 (d, J=10.9 Hz, 1H, H-19b), 4.15 (d, J=6.7 Hz, 2H,H-15), 4.52 (s, 1H, H-17a), 4.84 (s, 1H, H-17b), 5.39 (t, J=6.6 Hz, 1H,H-14).

6) ¹³C-NMR (CDCl₃, 125 MHz) δ 15.0 (C-20), 16.4 (C-16), 17.6 (C-2), 18.7(C-11), 21.8 (C-6), 24.2 (C-19), 35.4 (C-3), 38.0 (C-1), 38.1 (C-12),38.4 (C-10), 38.6 (C-7), 39.5 (C-4), 48.5 (C-5), 56.2 (C-9), 59.4(C-15), 72.0 (C-18), 105.5 (C-17), 123.1 (C-14), 140.5 (C-13), 148.3(C-8).

1) Physical property: White powder, M.P.=135.5˜136° C.

2) Polarity: [α]_(D) ²⁵-100° (c=0.24, CHCl₃)

3) Molecular weight: 222

4) Molecular weight: C₁₅H₂₆O

5) ¹H-NMR (CDCl₃, 300 MHz) δ 0.79 (d, J=6.9 Hz, 3H, H-12 or H-13), 0.87(d, J=6.6 Hz, 3H, H-13 or H-12), 1.09 (m, 1H), 1.28 (m, 1H), 1.27 (s,3H, H-15), 1.41-1.60 (m, 6H), 1.63 (s, 3H, H-14), 1.85-2.01 (m, 5H),5.51 (d like, J=4.2 Hz, 1H, H-7).

6) ¹³C-NMR (CDCl₃, 75 MHz) δ 15.3, 18.5, 21.5, 21.7, 23.6, 26.4, 27.9,31.1, 35.3, 36.8, 44.1, 45.4, 72.5 (C-2), 124.6 (C-7), 134.3 (C-6).

EXPERIMENTAL EXAMPLE 1 Investigation of Anti-Oxidative Activity of T.nucifera Extracts or Abietane Diterpenoid Compound Isolated from theSame of the Present Invention by TBARS Method

In order to investigate anti-oxidative activity to LDL of Torreyanucifera extract or abietane diterpenoid compound isolated from thesame, following experiments were performed.

Cu²⁺ is known to mediate LDL-oxidation. Thus, in the present invention,dialdehyde, which is an oxidation product of unsaturated fatty acidformed during Cu²⁺ mediated LDL-oxidation, was measured bythiobarbituric acid reactive substances (TBARS) method in order toinvestigate the LDL anti-oxidative activity of T. nucifera extracts orabietane diterpenoid compound separated from the same (Ahn, B. T. et.al., Low-density Lipoprotein-Antioxidant Constituents of SaururusChinensis, J. Nat. Prod. 64: 1562, 2001).

Centrifugation was performed with 300 ml of human blood plasma by usinga ultracentrifuge at 100,000×g for 24 hours to remove floatingVLDL/chylomicron layer in the upper part. The specific gravity of theremaining solution was adjusted to 1.063 g/ml. Centrifugation wasperformed again at 100,000×g for 24 hours, and 25 ml of the floating LDL(1.5˜2.5 mg protein/ml) in the upper layer was separated.

20 μl of the separated LDL (protein concentration, 50-100 g/ml) wasmixed with 210 μl of 10 μM phosphate-buffered saline (PBS), to which 10μl of T. nucifera extracts or abietane diterpenoid compound isolatedfrom the same was added respectively.

T. nucifera extracts or abietane diterpenoid compound isolated from thesame was dissolved in DMSO (dimethylsulfoxide), and the solution wasdiluted at different concentrations before use. Only a solvent was addedto a negative control, while probucol was added to a positive control.

10 μl of 0.25 mM CUSO₄ was added to the solution, followed by furtherreaction at 37° C. for 4 hours. Then, the reaction was quenched byadding 1 ml of 20% trichloroacetic acid (TCA). 1 ml of 0.67%thiobarbituric acid (TBA) solution dissolved in 0.05 N NaOH solution wasadded to the solution, followed by stirring for 10 seconds. The solutionwas heated at 95° C. for 5 minutes to induce coloring, then cooled downin ice water. Centrifugation was performed at 3000 rpm for 5 minutes toseparate supernatant. OD₅₄₀ was measured with a UV/VISspectrophotometer. Malondialdehyde (MDA) detected by the above coloringwas quantified.

PBS standard solution containing 0˜10 nmol MDA was prepared by 250 μlwith tetramethoxypropane malonaldehyde bis (dimethylacetal) storagesolution.

Coloring was also induced in the standard solution in analogy to theprocedure as described above and then OD₅₄₀ was measured to draw astandard curve of MDA.

MDA was quantified by using the standard curve in experiments with T.nucifera extracts or abietane diterpenoid compound isolated from thesame.

The results are shown in Table 1.

TABLE 1 Compound IC₅₀ (μM) T. nucifera leaves Hot water extracts 40% (4μg/ml) inhibition Methanol extracts 71% (4 μg/ml) inhibition T. nuciferaseeds Hot water extracts 35% (4 μg/ml) inhibition Methanol extracts 52%(4 μg/ml) inhibition

R = methylR = hydroxymethylR = aldehydeR = methylesterR =dimethoxymethyl 1.900.430.921.101.80 Probucol (Positive control) 1.55

AS shown in Table 1, IC₅₀ value of T. nucifera extracts (hot waterextracts and methanol extracts of leaves and seeds) of the presentinvention or abietane diterpenoid compounds isolated from the same wasvery low, indicating that the extracts has excellent anti-oxidativeactivity to LDL.

Therefore, T. nucifera extracts of the present invention or abietanediterpenoid compounds isolated from the same can be effectively used forthe prevention and the treatment of cardiovascular diseases includinghyperlipidemia and atherosclerosis which are caused by oxidation of LDL.

EXPERIMENTAL EXAMPLE 2 Effect on ACAT Activity of T. nucifera Extractsor Abietane Diterpenoid Compound or Terpenoid Compound Isolated from theSame of the Present Invention

Following experiments were performed to investigate the effect on ACATactivity of T. nucifera extracts or abietane diterpenoid compound orterpenoid compound isolated from the same of the present invention.

1. Preparation of ACAT Enzyme Sources

In order to investigate the effect on hACAT-1 and hACAT-2 activities,hACAT-1 and hACAT-2 proteins were first obtained by taking advantage ofbaculovirus expression system.

cDNAs of hACAT-1 and hACAT-2 obtained from human liver cDNA libraryscreening were inserted into baculovirus transfer vector, which was thenintroduced into insect sf9 cells to produce virus. Then, recombinantviruses of HACAT-1 and hACAT-2 were separated by plaque purificationmethod, and amplification was performed three times to increase titer ofviral stock. Hi5 insect cells having a good protein expression efficacywere infected with the recombinant virus, making multiplicity ofinfection as 1, followed by shaking culture at 27° C. for one day.

The cultured Hi5 cells over-expressing hACAT-1 and hACAT-2 werecentrifuged at 500×g for 15 minutes to separate microsomal fraction.Then, cells were crushed in hypotonic buffer by quick freezing quickthawing, followed by ultracentrifugation at 100,000×g for one hour.

The obtained microsomal fractions were suspended in storage buffersolution, adjusting protein content to 8 mg/ml, which was stored in adeep freezer before using.

2. Measurement of ACAT activity

6.67 μl of cholesterol dissolved in acetone at the concentration of 1mg/ml was mixed with 6 μl of 10% triton WR 1339 (Sigma Co.) in acetone.And acetone was evaporated by nitrogen gas. Distilled water was added tothe resultant mixture to adjust the content of cholesterol to 30 mg/ml.

To 10 μl of cholesterol solution was added 10 μl of 1 M KH₂PO₄ (pH 7.4),5 μl of 0.6 mM BSA (bovine serum albumin), 10 μl of the microsomalsolution obtained above, 10 μl of sample (T. nucifera extracts orabietane diterpenoid compound isolated from the same) and 45 μl ofdistilled water (total 90 μl). The mixture was pre-incubated in a 37° C.water bath for 30 minutes.

10 μl of [1-¹⁴C]oleyl-CoA solution (0.05 μCi, final concentration 10 μM)was added to the pre-incubated mixture, which was reacted in a 37° C.water bath for 30 minutes. To the mixture was added 500 μl ofisopropanol:heptane solution (4:1 (v/v)), 300 μl of heptane and 200 μlof 0.1 M KH₂PO₄ (pH 7.4), which was stirred vigorously with vortex andthen allowed to phase separation under gravity at room temperature for 2minutes.

The 200 μl of upper phase was put in a scintillation vial, to which 4 mlof scintillation solution (Lipoluma, Lumac Co.) was added. Radioactivityof the upper phase was measured by a scintillation counter (1450Microbeta liquid scintillation counter, Wallacoy, Finland).

ACAT activity was calculated based on the radioactivity, the amount ofsynthesized cholesteryl oleate, measured above and expressed as adefined unit, pico mole per 1 mg of protein for 1 minute (picomole/minute/mg protein).

The results are shown in Table 2.

TABLE 2 IC₅₀ (μM) Compound hACAT-1 hACAT-2 T. nucifera Hot water 65% 59%leaves extracts inhibition inhibition (100 μg/ml) Methanol 81% 77%extracts inhibition inhibition (100 μg/ml) T. nucifera Hot water 51% 46%seeds extracts inhibition inhibition (100 μg/ml) Methanol 41% 45%extracts inhibition inhibition (100 μg/ml)

R = methylR =hydroxymethylR = aldehydeR = methylesterR = dimethoxymethyl 46 74  37129 98  88 65  42309199 Formula 2 Dehydroabietinol  41  60Formula 3 Isopimaric acid 229 263 Formula 4 Kayadiol 120 155 Formula 5δ-cadinol  79 —

As shown in Table 2, T. nucifera extracts (hot water extracts andmethanol extracts of leaves and seeds) of the present invention orabietane diterpenoid compounds or terpenoid compounds isolated from thesame have excellent hACAT-1 and hACAT 2 inhibiting activities.

Therefore, T. nucifera extracts or abietane diterpenoid compounds orterpenoid compounds isolated from the same of the present invention canbe effectively used for the prevention and the treatment ofcardiovascular diseases including hyperlipidemia and atherosclerosiscaused by the synthesis and the accumulation of cholesteryl ester.

EXPERIMENTAL EXAMPLE 3 Cholesterol-Lowering Effect by T. nuciferaExtracts of the Present Invention

In order to investigate the blood cholesterol-lowering effect of T.nucifera extracts of the present invention, following experiments wereperformed.

30 specific pathogens free male C57BL/6J mice at the age of 6 weeks wereraised in an animal facility, in which temperature was maintained as22±3° C., humidity was kept as 55±10% and light was regulated as12L/12D. The mice were adapted for about one week before being used forexperiments. When they were at the age of 7 weeks (weight: 20˜22 g),they were divided into 5 groups according to randomized block design.Group 1 mice were given with high-cholesterol/high-fat diet (CRF-1;AIN-76 test animal's normal diet supplemented with 1.25% cholesterol and15% fat, Orient Yeast Co. Ltd., Japan), group 2 mice were given withhigh-fat/high-cholesterol (HFHC) diet together with 1% methanol extracts(wt/wt diet) of T. nucifera leaves (or 1% hot water extracts of T.nucifera leaves), and group 3 mice were treated with HFHC diet togetherwith 0.1% probucol (wt/wt diet). All the mice of every group were freeto take the diet and water for 10 days. Dietary intake was recordedeveryday and weights of animals were measured every 5 days. Uponcompletion of breeding, all the records were analyzed. As a result,there was no significant difference in dietary intake and in weightgaining among three groups, suggesting the normal growth in thosegroups.

All the mice in three groups were sacrificed 10 days after the beginningof animal tests, then their blood was examined.

Blood was taken from retro-orbital sinus of the sacrified mice byheparin treated capillary tube. Centrifugation was performed with bloodat 8,000×g for 10 minutes. Blood plasma was separated from supernatantand then stored in a deep freezer.

Total cholesterol content (TC) in blood plasma of each mouse ofexperimental group was measured by using a blood chemistry analyzer(CIBA Corning 550 Express, USA). In order to measure HDL-cholesterol,HDL measuring reagent (Chiron Diagnostics Co., USA), produced by themodified method for HDL measurement (references [{circle around (1)}Stein E. A., et al.: Development and evaluation of a method forquantitation of plasma high-density-lipoprotein cholesterol. Clin. Chem.24: 1112-1115, 1978; {circle around (2)} Finley P. R., et al.:Cholesterol in high-density lipoprotein: use of Mg²⁺/dextran sulfate inits enzymatic measurement. Clin. Chem., 24: 931-933, 1978; {circlearound (3)} Warnick G. R., et al.: Dextran sulfate-Mg²⁺ precipitationprocedure for quantitation of high-density lipoprotein cholesterol.Clin. Chem. 28: 1379-1388, 1982]) in which dextran sulfate and magnesiumsulfate were added to serum of each mouse to precipitate LDL and VLDLand then HDL in supernatant was measured, was mixed with blood plasma ofeach experimental group at the ratio of 1:10. The mixture was reacted ina 20˜25° C. incubator for 5 minutes, followed by centrifugation at8,000×g for 10 minutes. The resultant supernatant was put in a bloodchemistry analyzer. A statistical computer program (Microsoft Excel,Version 7.0) was used to investigate and confirm (student t-test) thesignificance in difference in the results of blood analysis amongexperimental groups.

The contents of total cholesterol and HDL-cholesterol are shown in Table3 and in Table 4.

TABLE 3 HFHC diet + methanol extracts HFHC diet + HFHC diet of T.nucifera probucol group leaves group group Total   207 ± 28.5 148.8 ±28.5 177.0 ± 33.7 cholesterol (mg/dl) HDL- 54.1 ± 6.0 39.2 ± 6.0 41.7 ±6.4 cholesterol (mg/dl) HDL/total 26.4 26.9 24.3 cholesterol (%)

TABLE 4 HFHC diet + hot water extracts HFHC diet + HFHC diet of T.nucifera probucol group leaves group group Total 204.5 ± 33.5 180.6 ±29.3 180.9 ± 32.6 cholesterol (mg/dl) HDL- 49.3 ± 6.6 43.7 ± 5.5 44.3 ±6.5 cholesterol (mg/dl) HDL/total 24.1 24.2 24.5 cholesterol (%)

As shown in Table 3, total cholesterol in plasma was 28.1% reduced inthe group treated with methanol extracts of T. nucifera leaves,comparing to a control. In the meantime, total plasma cholesterol in apositive control treated with probucol was 14.5% reduced, comparing to anegative control.

As shown in Table 4, plasma cholesterol was 11.7% reduced in the grouptreated with hot water extracts of T. nucifera leaves, comparing to acontrol. The total cholesterol in a positive control treated withprobucol was 11.5% reduced, comparing to a negative control.

The plasma HDL-cholesterol contents in T. nucifera extracts treatinggroup and in a positive control group treated with probucol were notmuch different from that of a negative control, indicating that theextracts reduces the plasma LDL-cholesterol.

Therefore, T. nucifera extracts of the present invention can beeffectively used for the prevention and the treatment of cardiovasculardisease by lowering blood cholesterol and LDL-cholesterol as well.

EXPERIMENTAL EXAMPLE 4 Acute Toxicity Test in Mice via OralAdministration

In order to investigate acute toxicity of T. nucifera extracts orabietane diterpenoid compound or terpenoid compound isolated from thesame, following experiments were performed.

12 of each specific pathogens free female and male ICR mice at the ageof 4 weeks (3 of each female and male/group) were raised in an animalfacility where temperature was regulated to 22±3° C., humidity wasadjusted to 55±10% and light was also regulated to 12L/12D. The animalswere adapted for one week before being used. Feed for laboratory animals(CJ Corp., Korea, for mouse and rat) and drinking water were provided atany time after being sterilized.

T. nucifera extracts prepared in the above example or abietanediterpenoid compound or terpenoid compound isolated from the same wasformulated by 50 mg/ml using 0.5% tween 80 as a solvent, then orallyadministered to mice at the concentration of 0.04 ml (100 mg/kg), 0.2 ml(500 mg/kg) and 0.4 ml (1,000 mg/kg) per 20 g of mouse body weight,respectively. Samples were administered orally just once. After theadministration, side effects and death were observed for 7 days.Precisely, changes of any symptoms and death of an animal were observed1 hour, 4 hours, 8 hours and 12 hours after the oral administration onthe first day, and once or more in the morning and once or more in theafternoon from the second day through the 7^(th) day.

On day 7, the animals were sacrificed and anatomized. The internalorgans were examined by the naked eye. From the day of oraladministration, weight changes were recorded everyday to investigatewhether or not the weights of animals were decreased by T. nuciferaextracts or abietane diterpenoid compound or terpenoid compound isolatedfrom the same.

As a result, neither specific clinical symptoms nor death by theadministration of the sample were observed in those animals. Inaddition, no toxicity was detected in mice either from the observationof weight changes, hematological tests, biochemical tests of blood, orautopsy.

Therefore, T. nucifera extracts, or abietane diterpenoid compounds orterpenoid compounds isolated from the same of the present invention areevaluated to be safe substance since it does not cause any toxic changein mice up to the level of 1,000 mg/kg and its estimated LD₅₀ value ismuch greater than 1,000 mg/kg in mice.

Preparative examples of the composition of the present invention aredescribed hereinafter.

PREPARATIVE EXAMPLE 1 Preparation of Pharmaceutical Compositions

Pharmaceutical compositions including T. nucifera extracts, or abietanediterpenoid compound or terpenoid compound isolated from the same of thepresent invention were prepared.

1. Preparation of powders Abietane diterpenoid compound of formula 1 2 gLactose 1 g

The above-mentioned ingredients were mixed together, and airtight bagwas filled with the mixture to prepare powders.

2. Preparation of tablets Abietane diterpenoid compound of formula 1 100mg Corn starch 100 mg Lactose 100 mg Magnesium stearate  2 mg

The above-mentioned ingredients were mixed together, and tablets wereprepared by tabletting according to the conventional tablet producingmethod.

3. Preparation of capsules Abietane diterpenoid compound of formula 1100 mg Corn starch 100 mg Lactose 100 mg Magnesium stearate  2 mg

The above-mentioned ingredients were mixed together, and gelatincapsules were filled with the mixture to prepare capsules according tothe conventional capsule producing method.

4. Preparation of injectable solutions Abietane diterpenoid compound offormula 1 10 μg/ml Dilute hydrochloric acid BP added to pH 3.5Injectable sodium chloride BP Maximum 1 ml

Abietane diterpenoid compound of formula 1 was dissolved in injectablesodium chloride BP, and pH of the solution was adjusted to 3.5 withdiluted hydrochloric acid BP. The volume was also adjusted withinjectable sodium chloride BP. After complete mixing, the solutionfilled 5 ml transparent glass type 1 ampules. By melting the glass, thesolution was air-tight sealed, which was autoclaved at 120° C. for 15minutes, resulting in the preparation of injectable solutions.

PREPARATIVE EXAMPLE 2 Preparation of Food

Food containing T. nucifera extracts, or abietane diterpenoid compoundor terpenoid compound isolated from the same of the present inventionwas prepared as follows.

1. Preparation of Spices and Condiments

Spices and condiments for health improvement that contains abietanediterpenoid compound of formula 1 by 0.2˜10 weight % were prepared.

2. Preparation of Tomato Ketchup and Source

Tomato ketchup and source for health improvement that contain abietanediterpenoid compound of formula 1 by 0.2˜1.0 weight % were prepared.

3. Preparation of Flour Foods

Abietane diterpenoid compound of formula 1 was added to flour by 0.1˜5.0weight %, and the mixture was used to prepare bread, cake, cookies,cracker and noodles to produce health improving foods.

4. Preparation of Soups and Gravies

Health improving processed meat, noodle soups and gravies were preparedby adding abietane diterpenoid compound of formula 1 by 0.1˜1.0 weight %to soups and gravies.

5. Preparation of Ground Beef

Health improving ground beef was prepared by adding abietane diterpenoidcompound of formula 1 by 10 weight % to ground beef.

6. Preparation of dairy products

Abietane diterpenoid compound of formula 1 was added to milk by 0.1˜1.0weight %, which was then used for the production of health improvingdairy products including butter and ice cream.

7. Preparation of Sun-Sik

Brown rice, barley, glutinous rice and Job's tears were gelatinized,dried and roasted by the conventional method, followed by pulverizationwith a pulverizer, resulting in 60-mesh granules.

Black bean, black sesame, Perilla japonica were steamed, dried androasted by the conventional method, followed by pulverization with apulverizer, resulting in 60-mesh granules.

Abietane diterpenoid compound of formula 1 was concentrated underreduced pressure in a vacuum concentrator, and then dried by spraydryer. The dried product was pulverized into 60-mesh granules.

Crops, seeds and the dried powder of abietane diterpenoid compound offormula 1 were mixed by the following ratio.

Crops (brown rice 30 weight %, job's tears 15 weight %, barley 20 weight%),

Seeds (Perilla japonica 7 weight %, black bean 8 weight %, black sesame7 weight %),

Dried powder of abietane diterpenoid compound of formula 1 (1 weight %),

Ganoderma lucidum (0.5 weight %),

Rehmannia glutinosa (0.5 weight %)

PREPARATIVE EXAMPLE 3 Preparation of Beverages

Beverages containing T. nucifera extracts, or abietane diterpenoidcompound or terpenoid compound isolated from the same of the presentinvention were prepared.

1. Preparation of Carbonated Beverage

Sugar (5˜10%), citric acid (0.05˜0.3%), caramel (0.005˜0.02%) andvitamin C (0.1˜1%) were mixed together, to which purified water (79˜94%)was added, resulting in syrup. The syrup was sterilized at 85˜98° C. for20˜180 seconds, then mixed with cooling water at the ratio of 1:4.Carbon dioxide was injected by 0.5˜0.82% thereto, resulting in thepreparation of carbonated beverage containing abietane diterpenoidcompound of formula 1.

2. Preparation of Health Beverage

Optional ingredients such as liquid fructose (0.5%), oligosaccharide(2%), sugar (2%), salt (0.5%), water (75%) and abietane diterpenoidcompound of formula 1 were mixed homogeneously. After pasteurization,the mixture was put in a small container such as pet or glass bottle,resulting in the preparation of health beverage.

3. Preparation of Vegetable Juice

0.5 g of abietane diterpenoid compound of formula 1 was added to 1,000ml of tomato or carrot juice to prepare health improving vegetablejuice.

4. Preparation of Fruit Juice

0.1 g of abietane diterpenoid compound of formula 1 was added to 1,000ml of apple or grape juice to prepare health improving fruit juice.

As explained hereinbefore, T. nucifera extracts, or abietane diterpenoidcompound or terpenoid compound isolated from the same of the presentinvention has excellent anti-oxidative activity to LDL and inhibits ACATactivity effectively. In addition, T. nucifera extracts of the presentinvention reduce blood LDL cholesterol and total cholesterol.

Therefore, the composition of the present invention can be effectivelyused for the prevention and the treatment of cardiovascular diseasesincluding hyperlipidemia and atherosclerosis caused by the LDL oxidationand the synthesis and accumulation of cholesteryl ester.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A method for preventing or treating a cardiovascular disease in a subject, comprising administering a pharmaceutically effective amount of Torreya nucifera extract to the subject.
 2. The method according to claim 1, wherein the cardiovascular disease is hyperlipidemia or atherosclerosis.
 3. The method according to claim 1, wherein the T. nucifera extract is prepared by extracting Torreya nucifera with water, alcohol or mixed solution thereof.
 4. The method according to claim 3, wherein the alcohol is methanol or ethanol.
 5. The method according to claim 3, wherein the Torreya nucifera comprises Torreya nucifera leaves, Torreya nucifera stems, or Torreya nucifera seeds.
 6. The method according to claim 1, wherein the Torreya nucifera extract is an ethyl acetate soluble extract of Torreya nucifera prepared by a process comprising the steps of extracting Torreya nucifera with water, alcohol or mixed solution thereof to obtain a crude extract; and fractionizing the water suspension of the crude extract with n-hexane, chloroform and ethyl acetate in that order, then to obtain a ethyl acetate soluble extract of Torreya nucifera.
 7. The method according to claim 1, wherein the Torreya nucifera extract comprises at least one of compounds represented by the following formula 1 to formula
 5.

(R is methyl, hydroxymethyl, aldehyde, methylester or dimethoxymethyl.)


8. A method for inhibiting an oxidation of low-density activity in a subject comprising administering a pharmaceutically effective amount of Torreya nucifera extract to the subject.
 9. The method according to claim 8, wherein the T. nucifera extract is prepared by extracting Torreya nucifera with water, alcohol or mixed solution thereof.
 10. The method according to claim 9, wherein the alcohol is methanol or ethanol.
 11. The method according to claim 9, wherein the T. nucifera comprises T. nucifera leaves, Torreya nucifera stems, or T. nucifera seeds.
 12. The method according to claim 8, wherein the Torreya nucifera extract is an ethyl acetate soluble extract of Torreya nucifera prepared by a process comprising the steps of extracting Torreya nucifera with water, alcohol or mixed solution thereof to obtain a crude extract; and fractionizing the water suspension of the crude extract with n-hexane, chloroform and ethyl acetate in that order, then to obtain a ethyl acetate soluble extract of Torreya nucifera.
 13. The method according to claim 8, wherein the Torreya nucifera extract comprises at least one of compounds represented by the following formula 1 to formula
 5.

(R is methyl, hydroxymethyl, aldehyde, methylester or dimethoxymethyl.)


14. A method for inhibiting Acyl-CoA:cholesterol acyltransferase (ACAT) in a subject comprising administering a pharmaceutically effective amount of Torreya nucifera extract to the subject.
 15. The method according to claim 14, wherein the T. nucifera extract is prepared by extracting Torreya nucifera with water, alcohol or mixed solution thereof.
 16. The method according to claim 15, wherein the alcohol is methanol or ethanol.
 17. The method according to claim 15, wherein the T. nucifera comprises T. nucifera leaves, Torreya nucifera stems, or T. nucifera seeds.
 18. The method according to claim 14, wherein the Torreya nucifera extract is an ethyl acetate soluble extract of Torreya nucifera prepared by a process comprising the steps of extracting Torreya nucifera with water, alcohol or mixed solution thereof to obtain a crude extract; and fractionizing the water suspension of the crude extract with n-hexane, chloroform and ethyl acetate in that order, then to obtain a ethyl acetate soluble extract of Torreya nucifera.
 19. The method according to claim 14, wherein the Torreya nucifera extract comprises at least one of compounds represented by the following formula 1 to formula
 5.

(R is methyl, hydroxymethyl, aldehyde, methylester or dimethoxymethyl.) 